Abstract

Chemotherapy-induced peripheral neuropathy is one of the most common dose-limiting side effects of cancer treatment. Currently, there is no Food and Drug Administration-approved treatment available. Histone deacetylase 6 (HDAC6) is a microtubule-associated deacetylase whose function includes regulation of α-tubulin-dependent intracellular mitochondrial transport. Here, we examined the effect of HDAC6 inhibition on established cisplatin-induced peripheral neuropathy. We used a novel HDAC6 inhibitor ACY-1083, which shows 260-fold selectivity towards HDAC6 vs other HDACs. Our results show that HDAC6 inhibition prevented cisplatin-induced mechanical allodynia, and also completely reversed already existing cisplatin-induced mechanical allodynia, spontaneous pain, and numbness. These findings were confirmed using the established HDAC6 inhibitor ACY-1215 (Ricolinostat), which is currently in clinical trials for cancer treatment. Mechanistically, treatment with the HDAC6 inhibitor increased α-tubulin acetylation in the peripheral nerve. In addition, HDAC6 inhibition restored the cisplatin-induced reduction in mitochondrial bioenergetics and mitochondrial content in the tibial nerve, indicating increased mitochondrial transport. At a later time point, dorsal root ganglion mitochondrial bioenergetics also improved. HDAC6 inhibition restored the loss of intraepidermal nerve fiber density in cisplatin-treated mice. Our results demonstrate that pharmacological inhibition of HDAC6 completely reverses all the hallmarks of established cisplatin-induced peripheral neuropathy by normalization of mitochondrial function in dorsal root ganglia and nerve, and restoration of intraepidermal innervation. These results are especially promising because one of the HDAC6 inhibitors tested here is currently in clinical trials as an add-on cancer therapy, highlighting the potential for a fast clinical translation of our findings.

(A) The inhibitory effects of ACY-1083 on the enzymatic activities
of HDAC1-HDAC9. The Y-axis represents the percentage of basal enzymatic activity
corresponding to various concentrations of ACY-1083. The curve fit was generated
by a 4-parameter, nonlinear regression in Graph Pad Prism. (B) The
effects of various concentrations of ACY-1083 on the acetylation level of the
HDAC6 substrate α-tubulin and on histone, which is not an HDAC6
substrate in SK-N-BE2 cells. (C) Structure and pharmacokinetics of
ACY-1083. ACY-1083 is present at biologically active concentrations 8 hours
after dosing in the plasma of mice received ACY-1083 i.p.
(n=3/group).

(A) Mice were administered with 2 rounds of cisplatin treatment; 3
days after the last cisplatin dose, mice received ACY-1083 (10 mg/kg) for 7 days
followed by 7 days rest and then another 7 days of cisplatin treatment.
Alternatively, mice received ACY-1083 (10 mg/kg) for 14 consecutive days.
Mechanical allodynia was measured using von Frey hairs, and the 50% paw
withdrawal threshold was calculated by the up–down method. Two-way
repeated-measure ANOVA revealed a main effect for time
(P<0.01), a group effect
(P<0.01), and a group-by-time interaction
(P<0.01). Tukey post-hoc analysis was used to
determine differences between groups at specified time points.
***P<0.001
between cisplatin + vehicle vs. saline + vehicle;
^^P<0.01 between cisplatin + ACY-1083 vs.
cisplatin + vehicle. n=6–14/group. (B) Mice
were administered with 2 rounds of cisplatin treatment; 3 days after the last
cisplatin dose, mice received ACY-1215 (30 mg/kg) orally for 14 days. Mechanical
allodynia was measured using von Frey hairs, and the 50% paw withdrawal
threshold was calculated by the up–down method. Two-way repeated-measure
ANOVA revealed a main effect for time (P<0.01), a group
effect (P<0.01), and a group-by-time interaction
(P<0.01). Tukey post-hoc analysis was used to
determine differences between groups at specified time points.
***P<0.001 between
cisplatin + vehicle vs. saline + vehicle;
ˆˆˆP<0.01 between cisplatin
+ ACY-1215 vs. cisplatin + vehicle.
n=6–8/group. (C) Spontaneous pain was measured
by the conditioned place preference test after 2 weeks of ACY-1083 treatment.
The Y-axis indicates the change in time spent in light chamber. Two-way ANOVA
revealed a signification interaction (P<0.05). Tukey
post-hoc analysis revealed significant differences between groups:
*P<0.05. n=6–8
mice/group. (D) Cisplatin-induced numbness was measured by the
adhesive removal test. Mice were tested in week 5 for cisplatin-induced
numbness. Statistical analysis using Two-way ANOVA revealed a significant
interaction (P<0.05). Tukey post-hoc analysis revealed
significant differences between groups:
*P<0.05.
n=4–10/group.

(A) Mitochondrial bioenergetics in DRG neurons from mice that had
received 2 rounds of cisplatin treatment and 11 injections of ACY-1083. Two-way
ANOVA revealed a signification difference for cisplatin + vehicle and
cisplatin + ACY-1083 versus saline + vehicle and saline
+ ACY-1083 for maximal respiratory capacity:
*P<0.05. (B) Mitochondrial
bioenergetics in DRG neurons from mice received 2 rounds of cisplatin and two
weeks of ACY-1083, tissues were taken two weeks after the last ACY-1083
treatment. Two-way ANOVA revealed a signification difference for cisplatin
+ vehicle versus all three other groups for maximal respiratory
capacity: *P<0.05.